The endocannabinoid 2-AG is highly susceptible to its hydrolysis into AA, which activates neutrophils through de novo LTB 4 biosynthesis, independently of CB activation. In this study, we show that 2-AG and AA stimulate neutrophils to release antimicrobial effectors. Supernatants of neutrophils activated with nanomolar concentrations of 2-AG and AA indeed inhibited the infectivity of HSV-1 and RSV. Additionally, the supernatants of 2-AG-and AA-stimulated neutrophils strongly impaired the growth of Escherichia coli and Staphylococcus aureus. This correlated with the release of a large amount (micrograms) of α-defensins, as well as a limited amount (nanograms) of LL-37. All the effects of AA and 2-AG mentioned above were prevented by inhibiting LTB 4 biosynthesis or by blocking BLT 1 . Importantly, neither CB 2 receptor agonists nor antagonists could mimic nor prevent the effects of 2-AG, respectively. In fact, qPCR data show that contaminating eosinophils express ~100-fold more CB 2 receptor mRNA than purified neutrophils, suggesting that CB 2 receptor expression by human neutrophils is limited and that contaminating eosinophils are likely responsible for the previously documented CB 2 expression by freshly isolated human neutrophils. The rapid conversion of 2-AG to AA and their subsequent metabolism into LTB 4 promote 2-AG and AA as multifunctional activators of neutrophils, mainly exerting their effects by activating the BLT 1 . Considering that nanomolar concentrations of AA or 2-AG were sufficient to impair viral infectivity, this suggests potential physiological roles for 2-AG and AA as regulators of host defense in vivo.

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are the main cytosolic sensors of single-stranded RNA viruses, including paramyxoviruses, and are required to initiate a quick and robust innate antiviral response. Despite different ligand-binding properties, the consensus view is that RIG-I and MDA5 trigger common signal(s) to activate interferon regulatory factor 3 (IRF-3) and NF-κB, and downstream antiviral and proinflammatory cytokine expression. Here, we performed a thorough analysis of the temporal involvement of RIG-I and MDA5 in the regulation of IRF-3 during respiratory syncytial virus (RSV) infection. Based on specific RNA interference-mediated knockdown of RIG-I and MDA5 in A549 cells, we confirmed that RIG-I is critical for the initiation of IRF-3 phosphorylation, dimerization and downstream gene expression. On the other hand, our experiments yielded the first evidence that knockdown of MDA5 leads to early ubiquitination and proteasomal degradation of active IRF-3. Conversely, ectopic expression of MDA5 prolonged RIG-I-induced IRF-3 activation. Altogether, we provide novel mechanistic insight into the temporal involvement of RIG-I and MDA5 in the innate antiviral response. While RIG-I is essential for initial IRF-3 activation, engagement of induced MDA5 is essential to prevent early degradation of IRF-3, thereby sustaining IRF-3-dependent antiviral gene expression. MDA5 plays a similar role during Sendai virus infection suggesting that this model is not restricted to RSV amongst paramyxoviruses.